The entire text of the above-referenced disclosure is specifically incorporated by reference herein without disclaimer.
The present invention relates generally to the fields of prophylaxis and treatment for viral infections. More particularly, it concerns the use of cyclin dependent kinase inhibitors for blocking replication of any DNA virus dependent on Cdk activity for proliferation, an example being herpesvirus and, more particularly, cytomegalovirus. Marek""s disease represents a chicken-CMV, equine abortion virus represents a horse variety and cattle have several specific CMV""s, to name but a few species-specific CMV""s.
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that infects greater than 80% of the human population. HCMV is capable of establishing a life-long infection following primary infection. Reactivation of HCMV often results during pregnancy, perfusion, and in immunocompromised states (Huang and Kowalik, 1993). HCMV rarely causes symptomatic disease in healthy immunocompetent individuals. However, HCMV can result in severe clinical manifestations in congenitally infected newborns and in immunocompromised individuals, such as those undergoing organ transplantation or those infected with HIV (Alford et al., 1990; Schooley, 1990; Rubin, 1990). Most animal species may be infected with species-specific cytomegalovirus.
HCMV is the most common cause of human congenital viral infections, occurring in approximately 1% of all new born infants (Weller, 1971; Alford et al., 1981). Congenital infections (e.g., those acquired during pregnancy) primarily result from reactivation of a latent infection in the mother and subsequent transmission to the fetus. The majority of HCMV infections are asymptomatic or subclinical. However, about 10% of infants infected in utero display clinical manifestations, approximately 5% display typical cytomegalic inclusion disease (CID), with the other 5% presenting atypical manifestations (Pass et al., 1980). Characteristic manifestations of CID include hepatomegaly, splenomegaly, microcephaly, jaundice, and petechiae (Weller and Hanshaw, 1964). The prognosis for congenital CID is bleak with the mortality rate reaching 30%. The surviving infants may suffer from severe mental and motor abnormalities (Alford et al., 1990).
Perinatal infections are acquired during or shortly after birth. It is estimated that 1 to 15% of infants born in the United States become infected with HCMV by 6 months of age (Alford et al., 1990). The majority of these, infections occur via ingestion of infected breast milk and contact with genital secretions during birth. Approximately 40-60% of infants breast-fed by sero-positive mothers and 25-50% of infants exposed to HCMV in the birth canal become infected (Stagno et al., 1980; Dworsky et al., 1983; Reynolds et al., 1973). The vast majority of these perinatal infections remain asymptomatic. However, there is evidence that perinatal HCMV infection may be associated with interstitial pneurmonitis and result in chronic lung disease (Brasfield et al, 1987).
HCMV""s ability to latently infect cells and become reactivated under immunosuppressed conditions pose a severe problem for AIDS patients (Chou, 1990; Drew et al., 1981; Quinn et al., 1987). Essentially 100% of HIV positive homosexual men present serological evidence of either recently acquired or reactivated HCMV infection (Collier et al,. 1987). According to Schooley (1990), HCMV can play at least four possible roles in the pathogenesis of AIDS. These include: 1) direct HCMV-induced morbidity associated with clinical symptoms, 2) increased immunosuppression induced by HIV, 3) enhanced HIV replication by trans-activation at the cellular level, and 4) destruction of the gastrointestinal mucosa, and predisposition to other infections.
HCMV infection is the primary cause of death in over 35% of AIDS patients (Gehrz, 1991). HCMV infection affects numerous organ systems including the central nervous system (CNS), pulmonary system, and gastrointestinal (GI) tract (Smith and. Brunnessel, 1994). HCMV infection of the CNS has been observed in more than 20% of AIDS patients at autopsy. Individuals with CNS infections present with clinically recognized neurological symptoms including encephalitis, polyradiculomyelitis, and neuropathy (Navia et al., 1986; Hawley et al., 1983; Said et al., 1991). It is estimated that xcx9c20% of AIDS patients will develop a gastrointestinal disease caused by HCMV (Dieterich et al., 1993). HCMV infection can result in lesions involving the oral mucosa, esophagus, intestine, and rectum (Kanas et al., 1987). The pain associated with these lesions of the GI tract can obstruct the intake of food and thus contribute to the already poor nutritional status of the patient (Villor et al., 1984).
HCMV also affects the immunological state of AIDS patients. HCMV induces numerous immunological abnormalities that can inhibit the patients ability to fight HIV and other infections. These abnormalities include; changes in the relative distribution of CD4 and CD8 T-lymphocytes (Drew et al., 1985; Detels et al., 1984), a decrease in release of and responsiveness to mitogens, (Kapasi and Rice, 1988; Sing and Garnett, 1984) altered HLA-DR expression, and altered antigen presentation (Fiala et al., 1993).
Interestingly, HCMV may also enhance HIV replication at a molecular level. Previous studies have demonstrated that HCMV and HIV can co-infect numerous cell types (Rice et al., 1984; Nelson et al., 1988). This coinfection of HCMV and HIV can result in the enhancement of HIV replication via the trans-activation of the long terminal repeat (LTR) sequences by HCMV regulatory elements (Schooley, 1990). Although the complete mechanism is far from clear, the net result is enhanced expression of the LTR and increased HIV replication.
A second group of individuals with increased risk of HCMV infection are organ transplant recipients. HCMV is the single most important infectious agent affecting organ recipients. At least two-thirds of these patients develop a HCMV-infection 1-4 months after transplantation (Rubin, 1990). According to Rubin (1990) there are three patterns of HCMV infection following organ transplantation; primary infection, reactivated infection, and superinfection. Primary infection has the greatest clinical impact and occurs when an individual who is sero-negative for HCMV becomes infected by a sero-positive donor organ or transfused blood. In reactivated infection the HCMV sero-positive recipient undergoes reactivation of endogenous latent virus. Virtually all organ recipients who are scro-positive for HCMV will show some evidence of HCMV reactivation (Rubin and Tolkoff-Rubin, 1984; Fiala et al., 1975). Finally, superinfection occurs when a sero-positive recipient receives an allograft from a sero-positive donor that is infected with a different HCMV strain.
HCMV-infection itself can result in a wide variety of conditions ranging from fevers to pneumonia and hepatitis (Rubin, 1990). Secondly, HCMV infection can produce an immunosuppressed state that exceeds that observed from the immunosuppressive drugs alone. This enhanced immunosuppression can result in increased opportunistic infections due to Pneumocystis carinii, fungi, and Listeria monocytogenes (Rubin, 1990). Finally, HCMV can directly contribute to the rejection of the allograft. These complications demonstrate the need to better understand the HCMV life cycle. Understanding the molecular mechanisms of HCMV replication should aid in the development of novel anti-virals to help control the spread and replication of HCMV.
The structure of the HCMV virion shares characteristics consistent with other DNA viruses, particularly the herpesviruses. The HCMV virion consists of four basic elements: 1) an electron-opaque core, 2) an icosahedral capsid surrounding the core, 3) a tegument surrounding the capsid, and 4) an envelope (Roizmnan and Sears, 1991).
HCMV gene expression is regulated by a complex interaction of both viral and cellular proteins (Huang and Kowalik, 1993). The most complex and well studied HCMV regulatory element is the major immediate early promoter (MIEP) which promotes transcription of the immediate early genes. The MIEP is an extremely strong and complex promoter containing numerous binding sites for cellular transcription factors that regulate IE gene expression (Boshart et al., 1985; Samnbuchetti et al., 1989). The MIEP contains various 17,18, 19, and 21 base pair repeats upstream of the transcriptional start site for IE1 and IE2. These repeats contain binding sites for cellular transcription factors such as NFxcexaB, SP1, NF1, and ATF/CREB. Other cis elements located outside the repeat elements include a serum-response element (SRE), glucocorticoid-response element (GRE), and AP-1 binding sites (reviewed in Huang and Kowalik, 1993). All of these cis-elements are thought to play important roles in regulating transcription from the MIEP. However, HCMV gene expression is not controlled exclusively by cellular transcription factors. The major immediate early proteins also play critical roles in regulating the MIEP. IE72 and 55 can upregulate transcription from the MIEP (Cherrington and Mocarski, 1989; Stenberg et al., 1990; Baracchini et al., 1992), whereas IE86 represses transcription from the MIEP (Stenberg et al., 1990). This repression by IE86 presumably occurs by direct or indirect interaction with a cis-repression signal in the MIEP that spans the transcriptional start site (Cherrington et al., 1991; Liu et al., 1991). Control of the MIEP by IE72, 55, and 86, is believed to provide an intermediate level of promoter activity necessary for maintaining proper levels of IE proteins for subsequent viral and cellular gene activation (Stenberg et al., 1990). HCMV early and late promoters are also regulated by IE proteins (Malone et al., 1990; Staprans et al., 1988). However, these promoters require both IE72 and IE86, along with cellular transcription factors for maximal expression (Chang et al., 1989; Depto and Stenberg, 1989; Malone et al., 1990). An example of this regulation occurs in transcription of the HCMV DNA polymerase gene. Transcription of the polymerase gene requires both IE72 and IE86 proteins together with the cellular transcription factor USF for promoter activity (Klucher et al., 1989). Immediate early proteins are also involved in activating cellular promoters. Recent studies have demonstrated HCMV""s ability to activate cellular genes such as, dihydrofolate reductase (Margolis et al., 1995), thymidine kinase (Estes and Huang, 1977), and topoisomerase II (Benson and Huang, 1990). Some of these promoters are regulated by interaction of IE proteins and cellular transcription factors (Margolis et al., 1995). The ability of HCMV to induce transcription and expression of these cellular genes is believed to be critical for successful viral replication.
The time required for maximal HCMV DNA synthesis is quite long compared to other herpesviruses. HCMV encodes its own DNA polymerase which is responsible for the synthesis of viral DNA. The HCMV DNA polymerase is a 140 kDa protein that possesses 3xe2x80x2 exonuclease activity (Huang, 1975a; Nishiyama et al., 1983). The HCMV polymerase is structurally and functionally distinct from cellular DNA polymerases and can be specifically inhibited by chemicals such as phosphonoacetic acid (Huang, 1975b).
HCMV DNA synthesis appears to occur in a biphasic manner, with synthesis beginning approximately 12 hrs post-infection. The first phase of synthesis slows at about 24 hr post-infection (Albrecht, 1989; Stinski, 1978). Maximum viral DNA synthesis, however, does not occur until 72-96 hr post-infection (Albrecht. 1989; Stinski, 1978). The reason for this slow rate of viral DNA synthesis is presently unknown. Some researchers have speculated that since HCMV stimulates host cell macromolecular synthesis, that at early times after infection when macromolecules are limiting, HCMV does not compete well with the host cell for these factor (Stinski, 1991). According to this hypothesis, the virus must first induce synthesis of macromolecules (i.e., nucleotides) before efficient viral DNA synthesis can occur.
The early events that one observes upon HCMV infection share many aspects in common with the cellular immediate early response that is provoked by addition of serum growth factors to serum-starved cells. The virus precipitates a very rapid increase in the intracellular concentration of many important second messengers (reviewed in Albrecht et al, 1989), including calcium, inositol trisphosphate, and diacylglycerol. Protein kinase C, along with phospholipase A2 are also activated following infection (AbuBakar et al., 1990). HCMV infection also induces dramatic changes in Na+/IK+ ATPase activity following infection (Albrecht et al., 1989). Albrecht (1989) has proposed that these changes in second messengers and intracellular ions are crucial for the morphological and pathological responses observed following HCMV infection.
HCMV replication in vivo occurs in terminally differentiated cells of epithelial and endothelial origin (Weller, 1971). Therefore, successful viral replication requires activation of the DNA synthetic machinery, as well as those pathways that are involved in biosynthesis of macromolecular precursors (nucleotides, polyamines, etc.). These processes are stringently repressed in post-mitotic cells. However, HCMV is capable of overcoming these restrictions to allow for viral replication. HCMV infection is capable of inducing numerous cellular genes involved in production of biosynthetic precursors and genes involved in transcriptional regulation. The expression of cellular immediate early genes such as c-fos, c-jun, and c-myc, all of which can act as transcription factors, are increased following HCMV infection (Boldogh et al., 1990; Monick et al., 1992; Colberg-Poley and Santomenna, 1988). Biosynthetic genes involved in nucleotide production such as dihydrofolate reductase, thymidine kinase, and ornithine decarboxylase are also induced following HCMV infection (Margolis et al., 1995; Estes and Huang, 1977; Isom, 1979). The induction of these genes demonstrates HCMV""s ability to overcome some of the restrains present in post-mitotic cells and induce the production of precursors required for HCMV replication.
It is generally agreed that in some cell types HCMV is capable of evoking a complete mitogenic response leading to replication of the host cell genome. (Albrecht et al., 1976; DeMarchi, 1983; Fumkawa et al., 1975; Kamiya et al., 1986). However, many (perhaps all) of these cells that exhibit a complete mitogenic response upon viral infection do not replicate the viral genome and fail to produce progeny virus (Albrecht et al., 1976; DeMarchi, 1983; Albrecht et al., 1989). The data suggest that failure to express the full complement of viral genes needed for HCMV replication may permit host cell DNA replication, such that a complete mitogenic response appears to be characteristic of cells that undergo abortive infection.
In cells that produce virus progeny (i.e., during productive infection), there is controversy concerning the extent to which the virus is capable of activating host cell DNA synthesis. It is clear that productively infected cells exhibit many manifestations of mitogenic stimulation (Albrecht et al., 1989; Yurochko et al., 1995; Bresnahan et al., 1996a; Wade et al., 1992; Estes and Huang, 1977; Jault et al., 1995). Some investigators have reported that complete (or nearly complete) replication of the cellular genome attends HCMV DNA synthesis in productively infected cells (Jault et al., 1995; St. Jeor and Hutt, 1977), leading to arrest of infected cells in a state that resembles G2 or M phase of the cell cycle (Jault et al., 1995). Other investigators have been unable to detect cellular DNA synthesis in cells that are replicating HCMV DNA and producing virus progeny (De Marchi, 1983; Albrecht et al., 1989, Bresnahan et al., 1996a). Thus, it is unclear to what extent HCMV is capable of eliciting a complete mitogenic response, leading to activation of the cellular DNA synthetic machinery and a significant increase in host cell DNA synthesis during the course of productive viral replication. Neither is it certain how the virus subverts the normal, post-mitotic constraints on DNA synthesis and biosynthesis of macromolecular precursors.
The ability of HCMV to activate cells is an important step in HCMV replication. However, the controversy of whether or not HCMV elicits a complete mitogenic signal and stimulates cellular DNA synthesis is ongoing. The cell cycle is divided into four distinct phases referred to as G1, S, G2 and M phase. These four phases encompass distinct molecular events that direct cell division. DNA synthesis and replication of the cellular genome occurs in S phase. The molecular events involved in chromosome separation and the formation of daughter cells occurs in M phase or mitosis (reviewed in Alberts et al., 1989). Gap 1 or G1 occurs after mitosis (cell division) and prior to the initiation of S phase. It is during this G1 phase that the cell will make a decision either to become quiescent or commit to another round of DNA replication (reviewed in Draetta, 1994; Sherr, 1994). Gap 2 or G2 represents the time between S phasc and the initiation of mitosis (Alberts et al., 1989). Recent studies have begun to elucidate the mechanisms involved in regulating progression through each of these phases. These mechanisms arc crucial for faithful replication of the genome and successful cell proliferation. Although the picture is far from clear some key genes regulating cell cycle progression have been identified.
Progression through the eukaryotic cell cycle is regulated by a family of serine/threonine protein kinases called cyclin-dependent kinases or Cdks (reviewed in Draetta, 1994; Sherr, 1993; Sherr, 1994). The activity of Cdks are closely regulated by numerous mechanisms some of which include, specific binding to regulatory subunits, called cyclins, binding to inhibitory subunits called cyclin kinase inhibitors (CKIs), phosphorylation, dephosphorylation, and protein degradation (reviewed in Elledge and Harper, 1994; Morgan, 1995; Sherr, 1994). The temporal activation and binding of cyclins to specific Cdks is shown in FIG. 1. The regulation and activity of Cdks is essential for successful cell cycle progression and cell proliferation.
Cdks comprise a family of at least 10-12 enzymes. Some of these enzymes have been studied in considerable detail. The first Cdk was identified in fission and budding yeast and designated cdc2 and cdc28, respectively (Nurse and Bissett, 1981; Lorincz and Reed, 1984). Cdk1 is the metazoan homologue of cdc2 and cdc28 (Hanks, 1987). Cdk1 complexes with both cyclins A and B and plays a critical role in regulating the G2/M phase transition (Sherr, 1983; Draetta, 1994). Cdk3 has been implicated in progression through G1 phase of the cell cycle (van den Heuvel and Harlow, 1993), but little more is known of this enzyme. Cdk5 functions in neural tissues (Lew et al., 1994). Cdk7 is the catalytic subunit of the Cdk activating kinase (CAK), which as the name implies, plays a role in regulating Cdk activity (Fesquet et al., 1993; Fisher and Morgan, 1994). Cdk2, Cdk4, and Cdk6 are thought to play critical roles in G1 phase progression. The precise details are far from clear, but it is generally believed that Cdk4 and Cdk6 regulate processes that are essential for progression through mid to late G1 phase; whereas Cdk2 regulates processes that are involved in the initiation of S phase (reviewed in Draetta, 1994; Sherr, 1993; Sherr, 1994). Cdk4 and Cdk6 are activated by association with one or another of the D-type (D1, D2, D3) cyclins (Bates et al., 1994; Matsushime et al., 1992; Meyerson and Harlow, 1994). Cdk2 is activated primarily by association with cyclin E or cyclin A (Dulic et al., 1992; Koff et al., 1992; Rosenblatt et al., 1992).
The biochemical consequences of cyclin binding to Cdks are not well understood, although it is known that binding of cyclins is a prerequisite for covalent modifications that are essential for catalytic activity (reviewed in Clarke, 1995; Morgan, 1995). For example, binding of an appropriate cyclin is required in order for Cdk-activating kinase (CAK) to phosphorylate T160 on Cdk2 and T174 on Cdk4 (Gu et al., 1993; Kato et a., 1994). The Cdks are inactive unless these carboxy-terminal threonine residues arc phosphorylated. Cyclin kinase inhibitors act in part to block CAK-dependent activation of Cdks (Gu et al., 1993; Kato et al., 1994; Slingerland et al., 1994), suggesting that cyclins and CKIs serve antagonistic functions with respect to CAK-dependent activation of Cdks.
Broadly speaking, there are two classes of metazoan CKIs. One class consists of the members of the INK family, which include but is probably not limited to p15, p 16, p18, and p19 (reviewed in Elledge and Harper, 1994; Hunter and Pines, 1994; Sherr and Roberts, 1995). INK type CKIs bind to and sequester Cdk4 and Cdk6, but not Cdk2. The sequestration of the Cdk subunit by these inhibitors prevents Cdk4 or 6 from complexing with its cyclin subunit (Serrano et al., 1993; Hannon and Beach, 1994). The INK type inhibitors also prevent Cdk4 and Cdk6 phosphorylation by dissociating the cyclin/Cdk binary complex which is the substrate for CAK. Activated cyclinD/Cdk4 complexes (i.e., those in which T174 has already been phosphorylated) are also inhibited due to displacement of the cyclin subunit by these inhibitors (reviewed in Elledge and Harper, 1994; Hunter and Pines, 1994; Sherr and Roberts, 1995).
The second family of CKIs, is comprised of Cip1 (also called WAF1, Cap20, and Sdi1) Kip1 and Kip2 (Harper et al., 1993; El-Deiry et al., 1993; Polyak et al., 1994; Toyoshima and Hunter, 1994). These CKIs bind to cyclin/Cdk binary complexes that contain Cdk2, Cdk3, Cdk4, or Cdk6 (reviewed in Elledge and Haper, 1994; Hunter and Pines, 1994; Sherr and Roberts, 1995). Unlike members of the INK family, Cip1 and Kip1 do not disrupt cyclin/Cdk complexes, but bind to such entities to form ternary and higher order complexes (Harper et al., 1993; Harper et al., 1995; Xiong et al., 1992, Zhang et al., 1993). Cip 1 and Kip1 are structurally related to each other, but not to the INK-type CKIs (Hannon and Beach, 1994; Polyak et al., 1994; Serrano et al., 1993; Toyoshima and Hunter, 1994). Both Cip1 and Kip1 have similar properties in vitro. Both inhibit phosphorylation by CAK at low stoichiometries, thereby preventing Cdk activation (Gu et al, 1993; Harper et al., 1993; Polyak et al., 1994; Toyoshima and Hunter, 1994). Cip1 appears to facilitate the formation of cyclin A/Cdk2 complexes at low concentrations of the inhibitor (Haper et al., 1995; Zhang et al., 1994), and it has been suggested that Cip1 may be involved in recruitment of cyclin A and Cdk2 to newly synthesized ternary complexes. At higher stoichiometries. Cip1 and Kip1 are potent inhibitors of activated Cdk2, Cdk3, Cdk4. and Cdk6 (Zhang et al., 1993: Gu et al., 1993; Harper et at. 1993). It is believed that Cip1 and Kip1 constitute an activation threshold for progression through the G1 phase of the cell cycle. According to this hypothesis. activation of Cdk2, Cdk4. and Cdk6 cannot occur until the abundance of the corresponding cyclin/Cdk complexes exceeds the concentration required to saturate the xe2x80x9cfreexe2x80x9d pools of Cip1 plus Kip1 (reviewed in Elledge and Harper, 1994; Hunter and Pines, 1994; Sherr and Roberts, 1995; Morgan, 1995).
The mechanism where by Cdks regulate cell cycle progression is complex and by no means complete. However, large amounts of data have been collected regarding how Cdks regulate transition from G1 into S phase. The majority of this work centers around the product of the retinoblastoma tumor suppressor gene, Rb. Rb functions in part by serving as a control point that connects extracellular signals and gene transcription. Rb is a phosphoprotein that is differentially phosphorylated throughout the cell cycle. During G0 or early G1, Rb is present in a hypophosphorylated form and exert its growth suppressive activity (reviewed in Weinberg, 1995; Nevins, 1992; Hinds and Weinberg, 1994). When Rb is present in this form it binds to and inactivates certain members of the E2F transcription factor family. The binding of Rb with E2F blocks E2F-mediated transcription of cellular genes that are required for entry into S phase, such as DNA polymerase ax, and dihydrofolate reductase (reviewed in Nevins, 1992; Farnham et al., 1993; Hinds and Weinberg 1994). G1 cyclin/Cdk complexes are believed to relieve this Rb-mediated suppression by phosphorylating Rb, which results in the release of E2F (Nevins, 1992; Weinberg, 1995). Upon its release E2F can then activate transcription of cellular genes essential for entry into S phase. A schematic of Cdk-mediated inactivation of Rb is shown in FIG. 2.
There is a substantial amount of data indicating that all G1 cyclin/Cdk complexes play critical roles in the initiation of and/or progression through S phase (reviewed in Draetta 1994; Elledge and Harper, 1995; Sherr, 1994), but until substrates besides Rb are found, the specific role for each cyclin/Cdk complex in regulating cell cycle progression will likely remain largely unknown. Anologous results are likely for other DNA viruses dependent on cell cycle stimulatory kinases for replication at least most other species-specfic CMV""s.
In vitro mammalian cell systems provide the most studied and best models for DNA virus infections. This is most spectacularly true for HCMV, where there is no accepted animal model. The well-studied in vitro HCMV systems provide a superior model for all mammalian DNA virus infections.
An important aspect of the present invention is a method for inhibiting proliferation of a DNA virus which is dependent upon events associated with cell proliferation for replication. The DNA virus include, for example any of the herpesvirus family such as Herpes simplex, e.g., and most particularly cytomegalovirus (especially human cytomegalovirus). The method involves administering a prophylactically or therapeutically effective amount of a cyclin-dependent kinase (e.g., Cdk2) inhibitor to a patient. The therapeutically effective amount of inhibitor is that amount sufficient to inhibit Cdk and therefore prevent viral replication. The most preferred inhibitor is roscovitine although olomoucine or other Cdk inhibitors are also acceptable. Once the knowledge of the present invention is available, many other Cdk inhibitors may be developed and these Cdk activity inhibitors will be useful for the therapy and prophylaxis of DNA viral infections. Herpesviruses such as herpes simplex, for example, are also treatable by the methods of the present invention. The preferred therapeutically or prophylactically effective amounts of the Cdk inhibitors of the present invention (e.g., roscovitine, olomoucine and the like) are about 0.1 xcexcg/kg body weight to about 1000 xcexcg/kg of body weight (more preferably 0.1 xcexcg/kg to 100 xcexcg/kg. However, more effective Cdk or Cdk2 inhibitors would have to be added at lesser concentrations. Likewise, weaker Cdk inhibitors would require greater amounts (i.e., 10-1000 xcexcg/kg). Other Cdk inhibitors may be readily developed and tested based upon the structures and assay methods described herein or well known to those of skill in the art.
Human cytomegalovirus is a herpesvirus that induces numerous cellular processes upon infection. Among these are activation of cyclin-dependent kinase 2 (Cdk2), which regulates cell cycle progression in G1 and S phase. Inhibition of cellular Cdk2 activity blocks HCMV replication. Inhibition of Cdk2 activity by roscovitine inhibits HCMV DNA synthesis, production of infectious progeny, and late antigen expression in infected cells in a dose-dependent manner. HCMV replication is also inhibited by the expression of a Cdk2 dominant negative mutant, whereas expression of wild type Cdk2 has no effect on viral replication. Activation of cellular Cdk2 is now found necessary for HCMV replication.
HCMV replication is particularly dependent on the activity of cyclin E-Cdk2. Drugs that inhibit the activity of Cdk2 profoundly inhibit virus replication. Drugs that block certain components in the proliferative activation of cells and their progression through the cell cycle also block HCMV replication. Other viruses, particularly DNA viruses and more particularly herpesviruses that are dependent on events activating cell proliferation for replication will be similarly inhibited.
As demonstrated herein. the administration of cytotoxically effective amounts of cdk2 inhibitors may also be used to selectively kill cells infected with a DNA virus such as HCMV, for example.
Administration of the Cdk inhibitors may be parenteral (intravascular, e.g.) enteral or topical (e.g., intracavitary). The topical intracavitary administration may be to any body cavity likely to be a scene for viral infection or to already possess infected cells.